HyPro-seq analysis of HeLa and ARPE-19 cells

The ability of RNAs to form specific contacts with other macromolecules provides an important mechanism for subcellular compartmentalization. We developed a suite of hybridization-proximity (HyPro) labeling technologies for unbiased discovery of proteins (HyPro-MS) and transcripts (HyPro-seq) associated with RNAs of interest in genetically unperturbed cells. To generate the HyPro-seq dataset reported here, fixed and permeabilized human cells were hybridized with digoxigenin-labeled oligonucleotide probes against noncoding RNAs 45S, NEAT1 or PNCTR and transcripts co-localizing with these RNAs were biotinylated in situ using a custom-engineered HyPro enzyme containing a digoxigenin-binding domain. Biotinylated RNAs were then purified and sequenced using the NextSeq 500 Illumina platform.